Constitutive mTOR activation in TSC mutants sensitizes cells to energy starvation and genomic damage via p53

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102117/1/emboj7601900.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102117/2/emboj7601900-sup-0001.pd

XBP1, Downstream of Blimp-1, Expands the Secretory Apparatus and Other Organelles, and Increases Protein Synthesis in Plasma Cell Differentiation

AbstractThe differentiation of B cells into immunoglobulin-secreting plasma cells is controlled by two transcription factors, Blimp-1 and XBP1. By gene expression profiling, we defined a set of genes whose induction during mouse plasmacytic differentiation is dependent on Blimp-1 and/or XBP1. Blimp-1-deficient B cells failed to upregulate most plasma cell-specific genes, including xbp1. Differentiating xbp1-deficient B cells induced Blimp-1 normally but failed to upregulate genes encoding many secretory pathway components. Conversely, ectopic expression of XBP1 induced a wide spectrum of secretory pathway genes and physically expanded the endoplasmic reticulum. In addition, XBP1 increased cell size, lysosome content, mitochondrial mass and function, ribosome numbers, and total protein synthesis. Thus, XBP1 coordinates diverse changes in cellular structure and function resulting in the characteristic phenotype of professional secretory cells

Epigenetic Activation of a Subset of mRNAs by eIF4E Explains Its Effects on Cell Proliferation

BACKGROUND: Translation deregulation is an important mechanism that causes aberrant cell growth, proliferation and survival. eIF4E, the mRNA 5′ cap-binding protein, plays a major role in translational control. To understand how eIF4E affects cell proliferation and survival, we studied mRNA targets that are translationally responsive to eIF4E. METHODOLOGY/PRINCIPAL FINDINGS: Microarray analysis of polysomal mRNA from an eIF4E-inducible NIH 3T3 cell line was performed. Inducible expression of eIF4E resulted in increased translation of defined sets of mRNAs. Many of the mRNAs are novel targets, including those that encode large- and small-subunit ribosomal proteins and cell growth-related factors. In addition, there was augmented translation of mRNAs encoding anti-apoptotic proteins, which conferred resistance to endoplasmic reticulum-mediated apoptosis. CONCLUSIONS/SIGNIFICANCE: Our results shed new light on the mechanisms by which eIF4E prevents apoptosis and transforms cells. Downregulation of eIF4E and its downstream targets is a potential therapeutic option for the development of novel anti-cancer drugs

Distinct mechanisms of translation of members of the mammalian elongation factor family, eEF1A-1 and eEF1A-2, during neuronal differentiation

Eukaryotic peptide elongation factor-1 alpha (eEF1A) is an abundantly expressed protein involved in the elongation step of protein synthesis. Its protein expression is regulated at the translational level, as its mRNA shifts from ribonucleoprotein (RNP) complexes to polyribosomes upon growth factor stimulation. The regulation of this shift is mediated by a terminal oligopyrimidine (TOP) element in the 5' untranslated region (UTR), of the eEF1A-1 mRNA. This shifting is also involved during differentiation and mammalian development. During development the expression of eEF1A-1 declines and eEF1A-2, a highly conserved homologue, becomes the dominant eEF1A isoform expressed in brain, heart, and skeletal muscle. In this thesis, three studies were undertaken to examine the regulation and involvement of eEF1A-1 and eEF1A-2 in protein synthesis in neurons. In the first study, the steady-state expression of eEF1A-1 and eEF1A-2 mRNAs and proteins was studied during retinoic acid-induced neuronal differentiation of P19 embryonic teratocarcinoma cells. The decline of eEF1A-1 and the increase in eEF1A-2 expression during neuronal differentiation were found to be regulated primarily at the transcriptional level, since there was a correlation between the levels of mRNA and protein expression. In differentiated P19 neurons translational repression of eEF1A-1 mRNA also contributed to the decrease of eEF1A-1 protein. This indicated that transcriptional and translational repression of eEF1A-1 mRNA occurred during neuronal differentiation. Unlike eEF1A-1, eEF1A-2 mRNA was associated primarily with polyribosomes, suggesting that eEFIA-2 is preferentially translated during P19 neuronal differentiation. In the second study, the eEF1A-1 isoform was shown to be preferentially synthesized in nerve growth factor (NGF) stimulated PC12 cells. The increased synthesis of eEF1A-1 was mediated at the translational level, as shown by the shifting of eEF1A-1 mRNA to polyribosomes after NGF stimul

siRNA mediated knockdown of eIF4E in NIH 3T3 cells.

<p>NIH 3T3 cells were transiently transfected with an siRNA against murine eIF4E or with a control siRNA, 4E-T-inv (scrambled sequence of human 4E-T), for 48 hr. Cells were lysed, and protein extracts were subjected to SDS-PAGE, followed by western blot analysis. The RNAi-mediated knockdown was repeated three times.</p

eIF4E and TOP mRNA translation.

<p>A) DNA segments encompassing the promoters and 5′ UTRs of L32, L32 mut (non-TOP), S16, S16 mut (non-TOP), L30 and β-actin were subcloned upstream of the firefly luciferase gene. 3T3-tTA-eIF4E cells were transfected with the various firefly luciferase reporters and a renilla luciferase reporter, which was used for transfection efficiency, and were cultured for 32 hr. Tetracycline containing medium was then replaced by a tetracycline free medium for 16 hr; control (non-induced) cells were cultured in parallel with tetracycline. Firefly luciferase activity (FLU) was measured and normalized against renilla luciferase activity (RLU). B) Luciferase activity of the reporters was measured in the parental cell line 3T3-tTA as described in (A). C) TOP sequences of L32, S16 and L30 are depicted. The arrows indicate the transcriptional start site. The nucleotide changes between L32 and S16 and L32 and L30 are underlined. D) Mutated reporters were generated by exchanging the TOP sequences of L32, S16 and L30. Luciferase assays were performed as described in (A). E) Luciferase activity of the reporters was measured in the parental cell line 3T3-tTA as described in (A). Assays were carried out in triplicate. Luciferase activities represent an average obtained from three independent experiments.</p